Journal: bioRxiv

Article Title: Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

doi: 10.1101/2024.09.21.614250

Figure Lengend Snippet: ( A ) A cartoon showing a cell-SLB assay for TIRF imaging of PD1:Shp2 association in Jurkat or human CD4 + T cells. ( B ) Left, representative TIRF images of the indicated mGFP-tagged PD1 variants (green) and anti-Shp2 (magenta) at the interface of a Jurkat cell and the SLB as depicted in A . Right, dot plots showing the anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). ( C ) Same as B , except that human CD4 + T cells expressing the indicated PD1 variants were imaged (n = 40 cells). ( D, E ) Same as A, B , except that DO11.10 cells were observed. ( F ) AA sequence alignment of hu PD1 ICD and mo PD1 ICD , with ITIM, PRS, PEQ/H, and ITSM highlighted. ( G ) Cartoon of a liposome reconstitution assay for measuring PD1 ICD :Shp2 tSH2 interaction. ( H ) Left, representative time courses of SC505 (Shp2 tSH2 ) F.I. at increasing concentrations of hu PD1 ICD . Right, % SC505 quenching 30 min after ATP addition plotted against the [ hu PD1 ICD ] and [ mo PD1 ICD ] (n = 3 independent experiments). ( I ) Bar graphs summarizing apparent K d of Shp2 tSH2 interaction with indicated PD1 ICD variants determined via assays shown in G and H (n = 3 independent experiments). ( J ) Left, representative TIRF images showing Shp2 recruitment to microclusters of indicated PD1 variants in a Jurkat-SLB assays. Right, dot plots showing anti-Shp2 F.I. normalized to PD1 F.I. (n = 40 cells). Scale bars: 5 µm. Data are mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant; student’s t-test.

Article Snippet: Snap-Cell505 star (SC505: #S9103S) was purchased from NEB.

Techniques: Imaging, Expressing, Sequencing, Reconstitution Assay